Biomedical subjects

V Bĕhal

Publications and source records attributed to V Bĕhal.

33 records · Page 2Linked to original sources

PubMed1987

Subcellular localization of enzymes in Streptomyces aureofaciens and its alteration by benzyl thiocyanate. I. Phosphatases and ATP-glucokinase.

Mycelia of a low- and a high-production strain of Streptomyces aureofaciens were converted into protoplasts and divided into five subcellular fractions in order to localize exopolyphosphatases (EC 3.6.1.11), triphosphatase (EC 3.6.1.25), inorganic diphosphatase (EC 3.6.1.1), apyrase (EC 3.6.1.5) and glucokinase (EC 2.7.1.2). The highest specific activity of enzymes hydrolyzing polyphosphates was found in cytoplasmic vesicles and membranes. Triphosphatase was detected in the periplasmic fraction. Periplasmic vesicles and cytoplasm exhibited a high activity of diphosphatase. Apyrase was found mainly in the fractions of membranes and cytoplasmic vesicles. Glucokinase was a cytoplasmic enzyme. The enzymes were released from membrane structures into cytoplasm or periplasmic space if benzyl thiocyanate (10 microM) was present in the growth medium.

Cations↗

PubMed1987

Subcellular localization of enzymes in Streptomyces aureofaciens and its alteration by benzyl thiocyanate. II. Anhydrotetracycline oxygenase and glucose-6-phosphate dehydrogenase.

The localization of anhydrotetracycline oxygenase and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was studied by determining the enzyme activities in subcellular fractions obtained by differential centrifugation of the mycelia of Streptomyces aureofaciens after lysozyme treatment. Glucose-6-phosphate dehydrogenase was a typical cytoplasmic enzyme both in the low- and high-production strain. Anhydrotetracycline oxygenase was found in the membrane fraction of the low-production strain. In the high-production strain, it was detected in several fractions, the highest activity being found in cytoplasm. The presence of 10 microM benzyl thiocyanate in the culture medium significantly changed the distribution of the latter enzyme in both strains. The redistribution of the enzymes is discussed with respect to tetracycline over-production.

Cell Fractionation↗

PubMed1986

Inhibition studies of glucose-6-phosphate dehydrogenase from tetracycline-producing Streptomyces aureofaciens.

NAD-linked activity of glucose-6-phosphate dehydrogenase from both low-producing and high-producing strains of Streptomyces aureofaciens was inhibited by ATP, ADP, AMP and Pi. The inhibition constants indicate that ADP was the most potent inhibitor. The NADP-linked activity remained unaffected even at relatively high concentrations of these inhibitors. All inhibitions of the NAD-linked activity were competitive with respect to NAD and noncompetitive with respect to glucose-6-phosphate. The results represent a possible new regulatory mechanism of glucose-6-phosphate dehydrogenase from a streptomycete and emphasize its involvement in the regulation of the biosynthesis of tetracyclines.

Adenine Nucleotides↗

PubMed1982

Effect of inorganic phosphate and benzyl thiocyanate on the activity of anhydrotetracyline oxygenase in Streptomyces aureofaciens.

The level of anhydrotetracyline oxygenase (an enzyme catalyzing the penultimate reaction in the biosynthesis of tetracyline) in Streptomyces aureofaciens was substantially influenced by the amount of inorganic phosphate and by the presence of benzyl thiocyanate in the cultivation medium. Phosphate decreased the specific activity of the enzyme, particularly when added to a growing culture. On the other hand, benzyl thiocyanate increased the specific activity of the enzyme. Its effect was most conspicuous in the growth phase. The effect of benzyl thiocyanate was more pronounced in the low-production strain than in the producing variant. Inorganic phosphate and benzyl thiocyanate did not influence the enzyme activity in vitro. Phosphate added to the growing cultures was readily absorbed by the cells. During this time the enzyme synthesis was repressed, derepresion occurred only after exhaustion of phosphate from the medium. The stimulatory effect of benzyl thiocyanate on the enzyme synthesis was not reversed by the inorganic phosphate added.

Oxygenases↗

PubMed1979

Synthesis and degradation of proteins and DNA in Streptomyces aureofaciens.

The rate of protein synthesis in Streptomyces aureofaciens, measured by incorporation of U-14C-L-leucine into cells, fluctuated during the production phase in the range of 10-15% of the values determined in the phase of intensive growth. Tetracycline partially inhibited the protein synthesis during the growth phase only. The proteins synthesized between the 6th and 18th hour of growth, were 75% degraded by the 48th hour. The DNA synthesis, measured by means of incorporation of 2-14C-thymine into the mycelium, occurred predominantly during the first 24 h of cultivation. Similarly, DNA synthesized between the 6th and 12th hour of cultivation was degraded by 75% after 48 h. The turnover of culture proteins is thus caused largely by degradation of old cells and growth of new ones which are more resistant to tetracycline. The activity of alanine aminotransferase and aspartate aminotransferase increase substantially towards the end of fermentation.

Alanine Transaminase↗

PubMed1979

[Action of chlortetracycline on the enzymes of the producer itself, Streptomyces aureofaciens].

When preparing a cell-free extract of Streptomyces aureofaciens from the culture to which chlortetracycline (1000 gamma/ml) was added before disintegration with alumina, a considerable decrease in the enzyme activity was reached. The presence of chlortetracycline during disintegration of the cells by means of glass beads did not substantially affect the enzyme activity of the cell-free extract. Addition of chlortetracycline directly to the reaction mixture for the enzyme assay (up to a concentration of 200 gamma/ml) did not induce any inhibition effect. The results suggested that during studying enzyme systems of organisms producing secondary metabolites, the product might distort the results on the use of an inconvenient method.

Cell Fractionation↗

PubMed1974